The long-term objectives of the proposed research program are to correlate the biochemical action of protein tyrosine kinases with cellular behavior. These enzymes play a crucial, but still often enigmatic role, in the signal transduction pathways responsible for cell growth and differentiation. Upon loss of key regulatory controls, either through over-expression or mutation, the oncogenic potential of these enzymes is released. Selective inhibitors, ligands, and sensors will be extraordinarily helpful in deciphering the role of these enzymes in specific pathways. In addition, inhibitors of kinase action may ultimately lay the foundation upon which therapeutically useful compounds can be devised. In particular, the primary focus of this research program deals with two key tyrosine kinases implicated in T cell activations. Consequently, inhibitors could furnish new therapies for autoimmune disorders, T cell derived cancers, and AIDs. The proposed research program seeks to expand upon our development of low nM affinity agents that target the SH1, SH2, and SH3 domains of tyrosine kinases, as well as sensors that allow us to fluorescently follow kinase activity. Specific aims include: (1) the acquisition of selective inhibitors/ligands that target the SH1, SH2, and SH3 domains of Fyn and Lck, (2) the preparation of selective fluorescent sensors of both enzymes, (3) the construction of caged inhibitors and sensors, and (4) the application of the chemical tools developed in aims 1 - 3 to explore the role of Fyn and Lck in T cell activation.